Long term primary adult rat hepatocyte cultures that simulate in vivo liver growth transitions will be used to study the regulation of alcohol dehydrogenase (ADH; E.C.1.1.1.1.). We will develop a radioimmunoassay that is specific for rat ADH and construct a radio-labeled DNA probe that is complementary (cDNA) to rat liver polysomal mRNA ADH. Both will be used to explore the molecular biology underlying coordinate changes in ADH activity and ethanol metabolism observed in the cultures during lag, logarithmic and stationary phases of growth. Other variables will be examined. These include possible alterations in protein turnover (to be measured by a "heavy"/"light" amino acid density-shift technique); hepatocyte growth rates and cell density (to be determined by standard growth measurements); extracellular conditioning factor or intracellular ADH-modifier levels (to be determined by appropriate media manipulations and mixing experiments); and ADH-structure per se (to be determined by polyacrylamide gel electrophoresis in dosium dodecyl sulfate and by isoelectric focusing). In addition, for the first time the direct effects of known and unknown ADH-activity modifying agents such as hormones (T3, steroids, prostaglandins, insulin, glucagon and EGF), nutrients (glucose) and substrate (ethanol) will be studied under chemically defined conditions, using highly differentiated mitogen-responsive stationary phase cultures. Results from these experiments should provide new insights into basic mechanisms that control alcohol metabolism in the liver and, moreover, shed light upon fundamental relationships between differentiation, and proliferative and developmental processes in normal epithelial cells.